Procedures for concentration and initial separation of TGF-alpha, EGF, and TGF-beta from human urine were developed using adsorption to microparticulate silica (Analytichem, Harbor City, CA, Cat. No. 11021) and fractional elution with increasing concentrations of acetonitrile. Further resolution was obtained by sequential chromatography based on molecular size (Bio-Gel), charge (CM-cellulose), and hydrophobicity (RP-HPLC). The high molecular weight (HMW) of 30,000 to 35,000 Mr, for TGF previously reported in the urine of various cancer patients is, in patients with malignant astrocytomas, indistinguishable from the HMW form of hEGF in terms of apparent molecular size, EGF receptor binding activity, EGF immunoreactivity and clonogenic activity. However, in comparison to bulk (25 liters) urine from normal individuals, equivalently large urine samples from these brain tumor patients contained about fourfold more HMW hTGF/hEGF. hTGF-alpha was not identified in either source of bulk urine. Secondly, in an in vitro study of HMW TGFs, using in part the above resolution scheme, conditioned medium from A673, a human rhabdomyosarcoma cell line, was found to contain principal peaks of EGF radioreceptor and clonogenic activity in SDS-PAGE slices corresponding to Mr 15,000 and 22,000 in an HPLC sample eluting at 25-26% acetonitrile, and two additional higher Mr activities in a 22-23% acetonitrile eluting region. Neither of these active regions from HPLC competed in radioimmunoassay under reduced and denatured conditions for hEGF or rTGF-alpha when using an antiserum raised against the 17 C-terminal amino acids of rTGF-alpha. These multiple forms of HMW hTGF-alpha produced in vitro suggest size heterogeneity and possible immunologic diversity among HMW members of the EGF/TGF-alpha family of growth promoting polypeptides. Thirdly, the urine of patients with disseminated breast cancer contains immunoreactive TGF-alpha which is not present in comparable control urine from normal individuals. Currently, the pre- and postoperative urine of patients with primary breast cancer without metastasis is being compared for TGF-alpha activity.